Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine relationship in patients with coronary artery diseases?

Department of Biochemistry, Ege University, Faculty of Medicine, Izmir, Turkey.
Clinical biochemistry (Impact Factor: 2.28). 06/2012; 45(16-17). DOI: 10.1016/j.clinbiochem.2012.06.024
Source: PubMed
ABSTRACT
Objectives:
Elevated homocysteine (Hcy) concentrations have been shown to be a risk factor for atherosclerotic vascular disease and thrombosis. Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of numerous conditions affecting the cardiovascular system. In this study, the influence of cardiovascular risk factors and other variables on Hcy and ADMA relationship in patients with coronary artery disease (CAD) was investigated.

Design and methods:
Seventy-five patients with CAD were divided into three tertiles according to their Hcy levels. The effect of age, gender, blood pressure, lipid profile, renal function, and the presence of diabetes, insulin resistance, heart failure, inflammation, overweight, smoking and severity of coronary atherosclerosis on Hcy and ADMA relationship was evaluated.

Results:
ADMA concentrations of patients in the middle and highest Hcy tertiles were significantly higher than the patients in the lowest tertile. When ADMA concentrations were adjusted for demographic, clinical and laboratory variables, the significant differences in ADMA concentrations between the tertiles were preserved. ADMA levels positively correlated with Hcy. Homocysteine levels positively correlated with serum creatinine and NT-proBNP concentrations and negatively correlated with glomerular filtration rates. Stepwise multiple regression analysis revealed Hcy as the unique predictor of ADMA levels.

Conclusion:
Homocysteine concentration has an effect on ADMA levels. There is a strong correlation between Hcy and ADMA. Cardiovascular risk factors do not have an influence on this relationship.

Full-text

Available from: Burcu Barutcuoglu
UNCORRECTED PROOF
1Q3 Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine
2 relationship in patients with coronary artery diseases?
3 Özben O.Q1 Işıklar
a,1
, Burcu Barutcuoğlu
b
, Ceyda Kabaroğlu
b,
,Işıl Mutaf
b
, Dilek Özmen
b
, Oya Bayındır
b
,
4 Mehdi Zoghi
c
, Hatice Uluer
d
5
a
Department of Biochemistry, Ege University, Faculty of Medicine, Izmir, Turkey
6
b
Department of Clinical Biochemistry, Ege University, Faculty of Medicine, Izmir, Turkey
7
c
Department of Cardiology, Ege University, Faculty of Medicine, Izmir, Turkey
8
d
Department of Biostatistics and Medical Informatics, Ege University, Faculty of Medicine, Izmir, Turkey
9
10
abstractarticle info
11 Article history:
12 Received 27 April 2012
13 Received in revised form 14 June 2012
14 Accepted 19 June 2012
15 Available online xxxx
1617
18
19
Keywords:
20 Atherosclerosis
21 Cardiovascular disease
22 Risk factors
23Objectives: Elevated homocysteine (Hcy) concentrations have been shown to be a risk factor for athero-
24sclerotic vascular disease and thrombosis. Increased asymmetric dimethylarginine (ADMA) levels have been
25implicated in the pathogenesis of numerous conditions affecting the cardiovascular system. In this study, the
26inuence of cardiovascular risk factors and other variables on Hcy and ADMA relationship in patients with
27coronary artery disease (CAD) was investigated.
28Design and methods: Seventy- ve patients with CAD were divided into three tertiles according to their
29Hcy levels. The effect of age, gender, blood pressure, lipid prole, renal function, and the presence of diabetes,
30insulin resistance, hea rt failure, inammation, overweight, smoking and severity of coronary atherosclerosis
31on Hcy and ADMA relationship was evaluated.
32Results: ADMA concentrations of patients in the middle and highest Hcy tertiles were signicantly higher
33than the patients in the lowest tertile. When ADMA concentrations were adjusted for demographic, clinical
34and laboratory variables, the signicant differences in ADMA concentrations between the tertiles were pre-
35served. ADMA levels positively correlated with Hcy. Homocysteine levels positively correlated with serum
36creatinine and NT-proBNP concentrations and negatively correlated with glomerular ltration rates. Step-
37wise multiple regression analysis revealed Hcy as the unique predictor of ADMA levels.
38Conclusion: Homocysteine concentration has an effect on ADMA levels. There is a strong correlation be-
39tween Hcy and ADMA. Cardiovascular risk factors do not have an inuence on this relationship.
40© 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
4142
43
44
45
Introduction
46 Elevated homocysteine (Hcy) concentrations have been shown to
47 be a risk factor for atherosclerotic vascular disease and thrombosis
48 [1,2]. Endothelial dysfunction as a consequence of increased oxidative
49 stress and impairments in bioavailable nitric oxide (NO) levels are
50 the main mechanisms underlying the vascular pathophysiology of
51 hyperhomocysteinemia [3]. Hcy induced endothelial dysfunction
52may involve an indirect adverse effect of increased asymmetric
53dimethylarginine (ADMA) production, an endogenous competitive in-
54hibitor of endothelial nitric oxide synthase (eNOS) [4,5]. ADMA is syn-
55thesized from L-arginine by arginine N-methyltransferases, which
56utilize S-adenosyl-L-methionine (an intermediate in the formation of
57Hcy from methionine) as a methyl donor, and hence Hcy and ADMA
58metabolism may be interrelated [6]. Furthermore, Hcy has been
59shown to increase generation of ADMA by inhibiting the expression
60and activity of dimethylarginine dimethylaminohydrolase (DDAH),
61which hydrolyzes ADMA to citrulline and dimethylamine [7].ADMA
62has an effect on NO concentrations directly by inhibiting NOS activity
63and/or by indirectly decreasing the available L-arginine concentration.
64ADMA has been shown to increase the oxidative stress by uncoupling
65the electron transport between NOS and L-arginine, and hence reduc-
66ing the generation and availability of endothelium-derived NO [8].
67In diet-induced hyperhomocysteinemic monkeys [9] and in
68humans with experimentally induced acute hyperhomocysteinemia
69[4], a concurrent increase in plasma ADMA levels has been shown to
70be associated with endothelial dysfunction. Thus, endothelial
Clinical Biochemistry xxx (2012) xxxxxx
Abbreviations: ADMA, asymmetric dimethylarginine; BMI, Body mass index;
CAD, coronary artery disease; DDAH, dimethylaminohydrolase; eGFR, estimated
glomerular ltration rate; ELISA, enzymelinked immunosorbent assay; eNOS, en-
dothelial nitric oxide synthase; Hcy, homocysteine; HDL, high density lipoprotein;
HOMA, homeostasis model assessment; LDL, low density lipoprotein; MDRD, Mod-
ication of Diet in Renal Disease Study; NO, nitric oxide; NT-proBNP, N-terminal pro
B-type natriuretic peptide; SAM, S-adenosyl-L-methionine.
Corresponding author.
E-mail address: ckabaroglu@gmail.com (C. Kabaroğlu).
1
This author currently works at the following address: Tavşanlı Devlet Hastanesi,
Klinik Biyokimya Laboratuvarı, Tavşanlı, Kütahya, Turkey.
CLB-08079; No. of pages: 6; 4C:
0009-9120/$ see front matter © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.clinbiochem.2012.06.024
Contents lists available at SciVerse ScienceDirect
Clinical Biochemistry
journal homepage: www.elsevier.com/locate/clinbiochem
Please cite this article as: Işıklar ÖO, et al, Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine relationship in
patients with coronary artery diseases?, Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.06.024
Page 1
UNCORRECTED PROOF
71 dysfunction observed in hyperhomocysteinemia may be secondary to
72 elevated ADMA levels and consequently to reduced eNOS activity.
73 Owing to the above mentioned metabolic associations, a relationship
74 between Hcy and ADMA may be expected in patients with cardiovas-
75 cular disease. Some clinical studies have demonstrated an association
76 between Hcy and ADMA levels [1013]. However, in some studies
77 conducted in patients with hyperhomocysteinemia [14], ischemic
78 heart disease [15] and post-methionine hyperhomocysteinemia
79 [16], type 2 diabetes with hyperhomocysteinemia [17], high vascular
80 risk [18] and peripheral vascular disease [19] no signicant relation-
81 ship was reported. Similar conicts have been reported in animal
82 models of hyperhomocysteinemia [20].
83 Increased ADMA levels have been implicated in the pathogenesis
84 of numerous conditions affecting the cardiovascular system, such as
85 hypercholesterolemia [21], hypertension [22], insulin resistance [23],
86 diabetes mellitus [24], renal failure [25], smoking [26] and overweight
87 [27]. These observations have raised questions about the impact of
88 such vascular risk factors on the relationship between Hcy and ADMA.
89 In this study, we aimed to investigate the inuence of different
90 factors including age, gender, diabetes, insulin resistance, blood pres-
91 sure, lipid prole, renal function, heart failure, inammation, over-
92 weight and smoking and the extent and severity of coronary
93 atherosclerosis on the relationship between Hcy and ADMA in the pa-
94 tients with coronary artery disease (CAD).
95 Material and methods
96 Patients
97 75 Turkish patients (mean age 58.4±9.8years, range 25 78 years)
98 with a cardiovascular disease who were hospitalized for coronary an-
99 giography (with at least one major epicardial coronary artery with
100 50% or more luminal obstructions) were involved in the study. The
101 medical history of each patient was obtained by a standardized ques-
102 tionnaire. The current and past health conditions and medications for
103 the last one year were recorded.
104 Non-smokers were dened as patients who had never smoked or had
105 notsmokedatleastforthelastve years. Smokers were dened as those
106 who had regularly smoked at least ve cigarettes per day for at least ve
107 years. Patients with intermediate smoking habits were not included.
108 The exclusion criteria were the presence of an acute coronary syn-
109 drome, systemic inammatory or primary liver diseases, renal failure
110 which required dialysis, renovascular or secondary hypertension or
111 malignant diseases. Individuals who had an angioplasty or a bypass
112 surgery, myocardial infarction or cerebrovascular events within the
113 last three months and vitamin B
12
or folic acid fortication within
114 the past year were excluded, also.
115 The patients were categorized by tertiles (lowest, middle and
116 highest) with regard to their Hcy concentrations.
117 The study was approved by the Research Ethics Committee of Fac-
118 ulty of Medicine, Ege University and a written informed consent was
119 obtained from each patient.
120 Blood sampling and laboratory analyses
121 Venous blood samples were drawn after a twelve hour overnight
122 fasting prior to the coronary angiography. Blood samples were cen-
123 trifuged at 3000g for 10 min within 30 min of collection. Routine bio-
124 chemical tests were analyzed on the blood collection day and the
125 samples for N-terminal pro B-type natriuretic peptide (NT-proBNP),
126 insulin and ADMA analyses were stored at 80 °C.
127 The serum ADMA concentration was measured by a competitive
128 ELISA method (DLD Diagnostika GmbH, Hamburg, Germany) on a
129 Dynex DSX Four Plate Automated ELISA Prosessing System (Dynex Tech-
130 nologies Chantilly, VA, USA) with a calibrator range between 0.1 and
131 5.0μmol/L. The lower detection limit was 0.05μmol/L. The intra-assay
132coefcients of variation were 15.4% at 0.82μmol/L and 12.8% at
1330.40μmol/L. Patient sample assays were performed in duplicates.
134The plasma Hcy concentrations were determined by a competitive
135chemiluminescence immunoassay method on an Immulite 2000 ana-
136lyzer (Siemens Medical Solutions Diagnostics, Los Angeles, USA). The
137inter-assay coefcients of variation were 10.6% at 9.4μmol/L. The
138lower detection limit was 2μmol/L.
139The serum NT-proBNP and insulin concentrations were determined
140by a sandwich chemiluminescence immunoassay method on a Hitachi
141Cobas e 411 analyzer (Roche Diagnostics, Mannheim, Germany). The
142detection limits were 0.06pmol/L and 0.2μU/mL for NT-proBNP and
143insulin, respectively. The intra-assay coefcients of variation were
1443.8% at 5.84pmol/L of NT-proBNP and 1.5% at 24.6μU/mL of insulin.
145Serum glucose, creatinine, total cholesterol, HDL-cholesterol,
146LDLcholesterol, triglyceride and high sensitive C-reactive protein
147measurements were carried out with routine laboratory methods
148on an automated analyzer (Modular, Roche Diagnostics, Mannheim,
149Germany) with commercially available kits.
150Denitions
151Body mass index (BMI) (kg/m
2
) was calculated as body weight/
152height
2
.
153The presence of insulin resistance was estimated according to the ho-
154meostasis model assessmen t (HOMA) score calculated with the following
155formula [28]; [fasting insulin (μU/mL)×fasting glucose (mmol/L)]/22.5.
156The glomerular ltration rate (GFR, mL/min/1.73m
2
) was estimat-
157ed according to the Modication of Diet in Renal Disease Study
158(MDRD) equation [29]; {186 (for men) or 138 (for women)×[serum
159creatinine (μmol/L)×0.0113]
1.154
×age (years)
0.203
}.
160The extent and severity of coronary atherosclerosis were quanti-
161ed by the Gensini scoring system [30].
162Statistical analysis
163For each variable, KolmogorovSimirnov test was applied to deter-
164mine the concordance to a Gaussian distribution. Variables with a
165Gaussian distribution were expressed as mean±SD, whereas variables
166without a Gaussian distribution were expressed as median (range).
167The categorical data were expressed as counts or percentages. The
168categorical variables were analyzed by a chi square analysis between
169the groups.
170One-way Analysis Of Variance (one way ANOVA) for parametric
171multiple comparison procedures, and Student t test for two indepen-
172dent group comparisons were used for variables with a Gaussian dis-
173tribution. For variables with a non-Gaussian distribution, Kruskal
174Wallis and Mann Whitney U tests were used. Dunnett's C and
175Bonferroni corrections were used where appropriate.
176Correlation coefcients were calculated either by Pearson's for
177parametric or by Spearman's for non-parametric data.
178Power analyses both for the whole group and the tertiles were
179conducted.
180Stepwise multiple linear regression analysis was used to identify
181those variables with the strongest associative inuence on ADMA.
182For the determination of multicollinearity, tolerance and variance in-
183ation factor (VIF) values of the independent varibales were evaluated.
184The level of statistical signicance was accepted as a p valueb 0.05.
185Data management was carried out with the statistical package pro-
186gram SPSS (Statistical Package for the Social Sciences Inc. Chicago IL,
187USA) version 17 for Windows.
188Results
189All of the subjects were grouped into three as tertiles of Hcy levels;
190lowest tertile (13.0μmol/L, n=25), middle tertile (13.116.9μmol/
191L, n=25) and highest tertile (17.0μmol/L, n=25).
2 Ö.O. Işıklar et al. / Clinical Biochemistry xxx (2012) xxxxxx
Please cite this article as: Işıklar ÖO, et al, Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine relationship in
patients with coronary artery diseases?, Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.06.024
Page 2
UNCORRECTED PROOF
192The demographic and clinical data of the patients are summarized
193in Table 1. The frequency of type 2 diabetes mellitus and the use of an
194oral anti-diabetic drug were signicantly different between the
195groups. No signicant differences were observed between the three
196groups with regard to other demographic and clinical data.
197The laboratory results of the patients in different Hcy tertiles are
198summarized in Table 2. ADMA concentrations of the middle and
199highest tertiles were signicantly higher than the lowest tertile
200(both pb 0.05), on the other hand, there was no statistically signi-
201cant difference between the middle and highest tertiles.
202The hyperhomocysteinemic patients had signicantly decreased e
203GFR levels compared with the patients in the lowest tertile. There was
204no statistically signicant difference neither between lowest vs. mid-
205dle nor highest vs. middle tertiles.
206Total cholesterol, HDL-cholesterol and LDL-cholesterol, triglycer-
207ides, fasting glucose, creatinine, C-reactive protein, NT-proBNP mea-
208surements and HOMA-IR indices were not different between the three
209groups.
210The mean ADMA concentrations for each Hcy tertile were rec-
211alculated after the Bonferroni or Dunnet C corrections which involved
212demographics, laboratory and clinical variables. The calculated ADMA
213concentrations were not signicantly different from the previous
214ones. The multiple comparisons of ADMA between Hcy tertiles were
215performed after the Bonferroni or Dunnet C corrections and the sta-
216tistical signicance was maintained at the same level.
Table 1t1:1
The demographic and clinical data of the patients in different homocysteine tertiles.
Q2
t1:2
t1:3 Demographics and
clinical data
Homocysteine tertiles
t1:4 Lowest tertile
(13.0μmol/L)
Middle tertile
(13.116.9μmol/L)
Highest tertile
(17.0μmol/L)
t1:5 Number of patients 25 25 25
t1:6 Age (years) 58.3±10.9 58.4±8.6 58.4±10.2
t1:7 Male/female (n, %) 21/4 (8416%) 21/4 (8416%) 24/1 (964%)
t1:8 BMI (kg/m
2
) 26.9±3.5 28.1±3.7 26.2±2.9
t1:9 Gensini score 25 (4178) 18 (2112) 39 (4122)
t1:10
t1:11 Blood pressure (mm/Hg)
t1:12 Systolic 120 (100175) 120 (100160) 120 (90160)
t1:13 Diastolic 80 (60100) 80 (60100) 80 (60100)
t1:14
t1:15 Patient History (n, %)
t1:16 AMI 7 (28%) 10 (40%) 12 (48%)
t1:17 Coronary angioplasty 6 (24%) 8 (32%) 7 (28%)
t1:18 Coronary bypass surgery 6 (24%) 4 (16%) 5 (20%)
t1:19 Peripheral arterial disease 1 (4%) 0 (0%) 1 (4%)
t1:20 Stroke 1 (4%) 1 (4%) 1 (4%)
t1:21 Type 2 diabetes mellitus
10 (40%) 5 (20%) 2 (8%)
t1:22 Smoking 6 (24%) 6 (24%) 7 (28%)
t1:23
t1:24 Medication (n/%)
t1:25 Oral anti-diabetics
10 (40%) 3 (12%) 2 (8%)
t1:26 Angiotensin converting
enzyme inhibitors
8 (32%) 8 (32%) 12 (48%)
t1:27 Angiotensin receptor
antagonists
8 (32%) 4 (16%) 5 (20%)
t1:28 Nitrates 6 (24%) 6 (24%) 7 (28%)
t1:29 Beta blockers 15 (60%) 10 (40%) 14 (56%)
t1:30 Calcium channel blockers 4 (16%) 5 (20%) 5 (20%)
t1:31 Diuretics 9 (36%) 10 (40%) 7 (28%)
t1:32 Trimetazidine 3 (12%) 4 (16%) 4 (16%)
t1:33 Anti-aggregative drugs 18 (72%) 16 (64%) 21 (84%)
t1:34 Statins 14 (56%) 14 (56%) 18 (72%)
Data variables with a Gaussian distribution are given as mean±SD, whereas variables
without a Gaussian distribution are expressed as median (range).
t1:35
Signicantly different between tertiles (p=0.024).t1:36
Signicantly different between tertiles (p=0.009).t1:37
Table 2t2:1
The laboratory results of the patients in different homocysteine tertiles.
t2:2
t2:3 Laboratory
parameters
Homocysteine tertiles
t2:4 Lowest tertile
(13.0μmol/L)
Middle tertile
(13.116.9μmol/L)
Highest tertile
(17.0μmol/L)
t2:5 Homocysteine
(μmol/L)
9.8±2.4 14.7±1.1
23.2±6.5
,
t2:6 ADMA (μmol/L) 0.55±0.17 0.68±0.06
0.75±0.17
t2:7 Total cholesterol
(mmol/L)
4.6±1.2 4.8±1.3 4.4±1.1
t2:8 HDL-cholesterol
(mmol/L)
1.2±0.3 1.1±0.3 1.1±0.3
t2:9 LDL-cholesterol
(mmol/L)
2.6±1.0 2.8±1.0 2.5±0.9
t2:10 Triglycerides
(mmol/L)
1.5 (0.36.6) 1.8 (0.63.6) 1.5 (0.64.5)
t2:11 Fasting glucose
(mmol/L)
6.4 (5.3210.64) 6.16 (5.0415.7) 5.82 (4.17.78)
t2:
12 HOMA-IR index 2.5 (0.812.6) 3.5 (0.8105.6) 2.6 (0.8 28.1)
t2:13 Creatinine (μmol/L) 88.4 (53.0114.9) 88.4 (61.9159.1) 97.1 (70.7185.6)
t2:14 MDRD-eGFR
(mL/min/1.73m
2
)
80.8±15.5 79.5±12.6 69.6±17.5
t2:15 C-reactive protein
(mg/L)
4(166) 5 (1169) 8 (136)
t2:16 NT-proBNP
(pmol/L)
2.08 (0.1442.78) 1.72 (0.3213.99) 2.66 (0.1853.74)
Data variables with a Gaussian distribution are given as mean±SD, whereas variables
without a Gaussian distribution are expressed as median (range).
t2:17
vs lowest tertile, pb 0.05.t2:18
vs middle tertile, pb 0.05.t2:19
Table 3 t3:1
The correlation coefcients between ADMA, homocysteine and relevant variables in all
the subjects. Correlation coefcients were calculated by Pearson's analysis for paramet-
ric and Spearman's analysis for non-parametric data.
t3:2
t3:3ADMA Homocysteine
t3:4ADMA 0.404
t3:5Age 0.108 0.018
t3:6BMI 0.087 0.080
t3:7Gensini score 0.067 0.062
t3:8Systolic blood pressure 0.022 0.130
t3:9Diastolic blood pressure 0.115 0.32
t3:10Total cholesterol 0.011 0.058
t3:11HDL-cholesterol 0.024 0.198
t3:12LDL-cholesterol 0.039 0.015
t3:13Triglycerides 0.056 0.007
t3:14Fasting glucose 0.159 0.189
t3:15HOMA-IR index 0.049 0.016
t3:16Creatinine 0.097 0.482
t3
:17MDRD-eGFR 0.120 0.399
t3:18C-reactive protein 0.028 0.090
t3:19NT-proBNP 0.095 0.267
pb 0.001. t3:20
pb 0.05. t3:21
Fig. 1. The correlation between homocyste ine and ADMA levels (r=0.404, p=0.0001).
3Ö.O. Işıklar et al. / Clinical Biochemistry xxx (2012) xxxxxx
Please cite this article as: Işıklar ÖO, et al, Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine relationship in
patients with coronary artery diseases?, Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.06.024
Page 3
UNCORRECTED PROOF
217 As shown in Table 3 and Fig. 1, taking into account all the subjects,
218 ADMA positively correlated with Hcy (r=0.404, pb 0.001). Hcy posi-
219 tively correlated with creatinine (r=0.482, pb 0.001) and NT-proBNP
220 (r=0.267, pb 0.05), whereas it negatively correlated with eGFR
221 (r=0.399, pb 0.001).
222 A stepwise multiple regression analysis was performed to deter-
223 mine the variables having the strongest impact on ADMA concentra-
224 tions. Age, gender, patient history, BMI, Gensini score, blood pressure,
225 Hcy, lipid prole, fasting glucose, HOMA-IR index, creatinine, e GFR
226 and NT-proBNP were accepted as independent variables. In the rst
227 step, homocysteine variable revealed 0.171 as the r
2
value which in-
228 dicated that approximately 17% of the variability in ADMA is
229 predicted by homocysteine (Table 4). The other variables were not
230 related to ADMA levels. We performed collinearity statistics and eval-
231 uated tolerance and VIF values. Tolerance values of the independent
232 variables were between 0.768 and 1.000. VIF values were between
233 1.000 and 1.302. There was not any collinearity among the indepen-
234 dent variables.
235 The study power was calculated as 0.96 by using the power anal-
236 ysis. The power analysis for the three study groups revealed a value
237 of 0.97.
238 Discussion
239 The present study investigated the interaction between Hcy and
240 ADMA levels in patients with CAD. We demonstrated a clear relation-
241 ship between Hcy and ADMA levels independent of other cardiovas-
242 cular risk factors.
243 Hyperhomocysteinemia has been shown to be a risk factor for the
244 development of atherosclerotic disease. The mechanisms of Hcy relat-
245 ed endothelial dysfunction seem to be multifactorial. Previous studies
246 demonstrated that the effects of Hcy are connected to its tendency to
247 form disulde bonds and to produce free oxygen radicals [31]. The
248 free oxygen radicals generated by Hcy react with NO and reduce its
249 bioactivity.
250 ADMA is derived from the breakdown of proteins that contain
251 methylated arginine residues in all cell types and NO synthesis is
252 inhibited by ADMA [4,5]. There are evidences that ADMA and Hcy
253 might be related: ADMA is correlated with Hcy in patients with stroke
254 and Hcy levels were suggested as the strongest predictor for ADMA
255 increase [12]. Similar associations between ADMA and Hcy were
256 found in studies including patients with Alzheimer's disease [13], ca-
257 rotid restenosis [32], systemic lupus erythematosus [33], early-onset
258 ischemic stroke [34], and coronary artery disease [35], also. Mamatha
259 et al. reported that Hcy levels and the presence of diabetes mellitus
260 were the best predictors of increased ADMA where Hcy explained
261 25% and diabetes mellitus explained 7% of ADMA concentration vari-
262 ance in a univariate analysis [34].
263 There are two possible links between Hcy and ADMA metabolism:
264 During the synthesis of Hcy from methionine via the demethylation
265 pathway, biosynthesis of ADMA occurs, which is linked to
266 S-adenosylmethionine. It was demonstrated that synthesis of ADMA
267 occurred via an S-adenosyl-L-methionine-dependent methylation of
268 L-arginine residues of proteins in cultured human endothelial cells
269 [6]. Another possible link is the inhibition of DDAH enzyme activity
270by Hcy. DDAH activity is critical in regulating ADMA levels. DDAH
271contains 4 cysteine groups in its amino acid sequence. The sulfhydryl
272groups in these groups play an important role for its enzyme function,
273since SH-blocking agents such as p-chloromercuribenzoat, HgCl
2
and
274Hcy act as potent inhibitors of DDAH [36]. Stuhlinger et al. have dem-
275onstrated that Hcy had an oxidative effect on DDAH, forming a disul-
276de bond with the sulfhydryl group in the catalytic site. Hcy bounds
277directly to a recombinant DDAH to inhibit its activity [37]. In addition
278to DDAH sensitivity to oxidative stress, this sulfhydryl moiety in the
279catalytic site can be nitrosylated, which causes inhibition of DDAH
280[38].
281This opinion is further supported by the structural analysis of the
282DDAH enzyme demonstrating that the S atom of Cys-249 in the active
283site of the enzyme might be vulnerable to oxidation and nitrosylation
284[39]. Therefore, researchers speculated that inhibition of DDAH in
285hyperhomocysteinemia might be due to the nucleophilic attack of
286the sulfur atom of Cys-249 by Hcy. These ndings suggest that eleva-
287tion of ADMA in hyperhomocysteinemia depends on interference of
288Hcy with DDAH rather than increased methylation via increases of
289methionine and SAM.
290Plasma ADMA levels were found to be elevated in subjects with
291metabolic disorders associated with atherosclerosis and endothelial
292dysfunction, such as renal failure [25], essential hypertension [22], in-
293sulin resistance [23], diabetes [24], and hyperlipidemia [40]. For the
294elimination of the confounding factors, we investigated the relation-
295ship between ADMA and situations involved in the development of
296endothelial dysfunction. Stepwise multiple regression analysis
297showed that Hcy was the unique predictor of ADMA. Contrary to
298some studies [26,41] we did not nd any relationship between eGFR
299and ADMA. Kielstein et al. reported that glomerular ltration of
300ADMA is markedly low because of the signicant protein binding
301property of this substance, and elevation of ADMA levels was inde-
302pendent of the type of renal disease [42]. DDAH which hydrolyses
303ADMA is highly expressed in the kidney [36]. The mechanism of in-
304creased ADMA concentrations in patients with renal disease is more
305likely to be the impairment in ADMA degradation by renal DDAH
306rather than reduced renal excretion. None of the patients in our
307study had end-stage renal disease. In contrast to previous studies
308showing ADMA elevations in patients with diabetes [24], hyperten-
309sion [22] or hypercholesterolemia [21] no major inuence of such
310risk factors was found in ADMA levels in our patients with CAD.
311These ndings are in agreement with a study conducted in a Swedish
312population with cerebrovascular diseases reporting no association
313between ADMA levels and any of the risk factors [43].
314The relationship between LDL-cholesterol and ADMA is unclear.
315Boger et al. found an association between ADMA and LDL-cholesterol
316[21]. However, there are conicting reports regarding the relationship
317between ADMA and LDL-cholesterol [44]. We could not nd any asso-
318ciation between increased ADMA levels and smoking which has been
319described in some studies [26,45]. The relationship between smoking
320and ADMA remains controversial [27]. Hilde et al. found strong rela-
321tionship between BMI and plasma levels of ADMA [27]. We did not de-
322tect such an association; however variations in BMI across the usually
323observed ranges might have not markedly affected ADMA levels.
324Observations in experimental [46] and clinical [47] studies suggest
325that plasma ADMA levels are increased in a congestive heart failure.
326This situation was explained by the compensatory role of a circulating
327endogenous NO synthase inhibitor against induced NO synthase
328activity seen in patients with a heart failure. To establish the effect
329of a heart failure on ADMA we evaluated the pro-BNP levels of CAD
330patients. There was no relation between ADMA and pro-BNP. In one
331study, it was reported that plasma ADMA level correlated signicant-
332ly with the extent and severity of coronary atherosclerosis as assessed
333by coronary angiography [48], but in another study there was no sig-
334nicant correlation [35]. In our study, ADMA levels of patients with
335CAD did not correlate with the severity coronary atherosclerosis.
Table 4t4:1
In the step-wise multiple regression analysis, keeping ADMA as the dependent factor,
age, gender, BMI, Gensini score, blood pressure, patient history, medication, homocys-
teine, lipid prole, glucose, HOMA-IR index, creatinine, MDRD-e GFR and NT-proBNP
variables were entered into the model as independent variables. Except homocysteine,
all the other variables were excluded in the rst step.
t4:2
t4:3 Dependent variable Beta Standard error of beta t p-Value
t4:4 Constant 0.496 0.045 11.042 b0.0001
t4:5 Homocysteine 0.01 0.003 3.779 b0.0001
4 Ö.O. Işıklar et al. / Clinical Biochemistry xxx (2012) xxxxxx
Please cite this article as: Işıklar ÖO, et al, Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine relationship in
patients with coronary artery diseases?, Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.06.024
Page 4
UNCORRECTED PROOF
336 In conclusion, this present study demonstrated that except Hcy,
337 the cardiovascular risk factors and some other variables had no effect
338 on ADMA levels. After adjustments of demographic, clinical and labo-
339 ratory variables, no effect was observed on the relationship between
340 Hcy and ADMA. These results suggest that plasma Hcy level is the
341 major factor which affects plasma ADMA concentrations in patients
342 with CAD and interventions for lowering homocysteine concentra-
343 tions may also induce a decrease in ADMA levels.
344 Conict of interest
345 Authors declare no conict of interest.
346 Acknowledgment
347 None.
348 Funding
349 This work was supported by the Ege University Research Projects,
350 Izmir, Turkey.
351 Author contributions
352 Ozben O. Isiklar: conduct and design of the study, data collection
353 and analysis.
354 Burcu Barutcuoglu : data interpretation and manuscript writing.
355 Ceyda Kabaroglu: data interpretation and manuscript writing
356 Isil Mutaf: conduct and design of the study, data analysis and in-
357 terpretation, manuscript writing
358 Dilek Ozmen: data interpretation and manuscript writing
359 Oya Bayindir: conduct and design of the study
360 Mehdi Zoghi: coronary angiography evaluations and patient
361 recruitment
362 Hatice Uluer: statistical analysis
363Q4
Q
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6 Ö.O. Işıklar et al. / Clinical Biochemistry xxx (2012) xxxxxx
Please cite this article as: Işıklar ÖO, et al, Do cardiac risk factors affect the homocysteine and asymmetric dimethylarginine relationship in
patients with coronary artery diseases?, Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.06.024
Page 6
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    • "Second, Isiklar et al. [41] have recently observed the lack of correlation between ADMA and Gensini score in 75 stable angina subjects, including 17 type 2 diabetics. Nevertheless, ADMA was unrelated to the degree of IR in the both cited studies [35,41]. Therefore, in order to differentiate between the effects of ADMA and IR on coronary atherosclerosis, we investigated mutual relations between plasma ADMA, the degree of IR and angiographic indices of CAD extent and severity in non-diabetic men with stable CAD. "
    [Show abstract] [Hide abstract] ABSTRACT: Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthesis inhibitor, and insulin resistance (IR) have been implicated in atherogenesis. Our aim was to estimate relations between ADMA, the magnitude of IR and angiographic indices of extent and severity of coronary atherosclerosis in non-diabetic men with stable coronary artery disease (CAD). We studied 151 non-diabetic men (mean age 57 +/- 11 years) with stable angina, obstructive CAD (at least 1 luminal diameter stenosis of >=70% in major coronary segments) and without heart failure, and 34 age-matched controls free of >=50% coronary narrowings. The following CAD indices were computed: the number of major epicardial vessels with >=70% stenosis, Sullivan extent score representing a proportion of the visible coronary tree with vessel wall irregularities, and Gensini score which reflects both CAD severity and extent, yet assigning a heavier weight to proximal segments and to the more severe narrowings by a non-linear point system. An estimate of IR was derived by homeostasis model assessment (HOMA-IR) from fasting insulin and glucose. Among the CAD patients, the proportions of subjects with 1-vessel, 2- vessel and 3-vessel CAD were 26%, 25% and 49%, respectively. ADMA levels were higher in patients with obstructive CAD compared to the controls (0.51 +/- 0.10 vs. 0.46 +/- 0.09 mumol/L [SD], P = 0.01), whereas HOMA-IR was similar (median, 3.2 [interquartile range: 2.4--4.9] vs. 2.9 [2.3--4.7], P = 0.2). Within the CAD group, ADMA increased across ascending quartiles of Sullivan score (Spearman's rho = 0.23, P = 0.004), but not with Gensini score (rho = 0.12, P = 0.15) or the number of vessels involved (rho = 0.08, P = 0.3). ADMA correlated to log-transformed Sullivan score (r = 0.21, P = 0.008), which was only slightly attenuated upon multivariate adjustment (beta = 0.19 +/- 0.08 [SEM], P = 0.015). HOMA-IR did not differ according to any measure of angiographic CAD (P >= 0.2). ADMA and log(HOMA-IR) were mutually unrelated (r = 0.07, P = 0.4). ADMA is associated with diffuse but not focal coronary atherosclerosis in non-diabetic men with stable CAD irrespectively of the degree of IR. The independent relationship between ADMA and coronary atherosclerotic burden may contribute to the well-recognized prognostic effect of ADMA in CAD.
    Full-text · Article · Oct 2013 · Cardiovascular Diabetology
  • [Show abstract] [Hide abstract] ABSTRACT: Background/Aims: Homocysteine-induced endothelial dysfunction favors the development of cardiovascular diseases through accumulation of endogenous nitric oxide (NO) synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) is the major enzyme for the degradation of ADMA in endothelial cells. The purpose of this study was to determine whether suppressed DDAH2 expression contributed to impairments of DDAH/ADMA/NOS/NO pathway induced by homocysteine in endothelial cells and whether DDAH2 overexpression could prevent endothelial cell dysfunction caused by homocysteine. Methods: Liposome-mediated transfection of endothelial cells was performed to establish the cell line of DDAH2 overexpression. After treatment of cells with 1 mmol/L homocysteine for 24 h, the transcription and expression of DDAH1 and DDAH2, DDAH and NOS activities as well as ADMA and NO concentrations were measured. Results : Treatment of endothelial cells with homocysteine significantly suppressed the transcription and expression of DDAH2 but not DDAH1. This suppression was associated with the declined DDAH activity, increased ADMA accumulation, inhibited NOS activity and decreased NO production in endothelial cells. DDAH2 overexpression not only resisted homocysteine-induced decline of DDAH activity, but also decreased the accumulation of endogenous ADMA, subsequently attenuated the reductions of NOS activity and NO production induced by homocysteine. Conclusions: These results indicate that suppression of DDAH2 expression is a culprit for homocysteine-induced impairments of DDAH/ADMA/NOS/NO pathway in endothelial cells, and therapeutic manipulation of DDAH2 expression may be a promising strategy for preventing endothelial dysfunction and cardiovascular diseases associated with hyperhomocysteinemia.
    No preview · Article · Nov 2012 · Cellular Physiology and Biochemistry
  • [Show abstract] [Hide abstract] ABSTRACT: Homocystinuria is a neurometabolic disease caused by a severe deficiency of cystathionine beta-synthase activity, resulting in severe hyperhomocysteinemia. Affected patients present several symptoms including a variable degree of motor dysfunction. In this study, we investigated the effect of chronic hyperhomocysteinemia on the cell viability of the mitochondrion, as well as on some parameters of energy metabolism, such as glucose oxidation and activities of pyruvate kinase, citrate synthase, isocitrate dehydrogenase, malate dehydrogenase, respiratory chain complexes and creatine kinase in gastrocnemius rat skeletal muscle. We also evaluated the effect of creatine on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 µmol/g body weight) and/or creatine (50 mg/kg body weight) from the 6th to the 28th days of age. The animals were decapitated 12 h after the last injection. Homocysteine decreased the cell viability of the mitochondrion and the activities of pyruvate kinase and creatine kinase. Succinate dehydrogenase was increased other evaluated parameters were not changed by this amino acid. Creatine, when combined with homocysteine, prevented or caused a synergistic effect on some changes provoked by this amino acid. Creatine per se or creatine plus homocysteine altered glucose oxidation. These findings provide insights into the mechanisms by which homocysteine exerts its effects on skeletal muscle function, more studies are needed to elucidate them. Although creatine prevents some alterations caused by homocysteine, it should be used with caution, mainly in healthy individuals because it could change the homeostasis of normal physiological functions. Copyright © 2012 John Wiley & Sons, Ltd.
    No preview · Article · Dec 2012 · Cell Biochemistry and Function
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